Antiviral responses in a Jamaican fruit bat intestinal organoid model of SARS-CoV-2 infection

Bats are natural reservoirs for several zoonotic viruses, potentially due to an enhanced capacity to control viral infection. However, the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB, Artibeus jamaicensis) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Upon infection with SARS-CoV-2, increased viral RNA and subgenomic RNA was detected, but no infectious virus was released, indicating that JFB organoids support only limited viral replication but not viral reproduction. SARS-CoV-2 replication was associated with significantly increased gene expression of type I interferons and inflammatory cytokines. Interestingly, SARS-CoV-2 also caused enhanced formation and growth of JFB organoids. Proteomics revealed an increase in inflammatory signaling, cell turnover, cell repair, and SARS-CoV-2 infection pathways. Collectively, our findings suggest that primary JFB intestinal epithelial cells mount successful antiviral interferon responses and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways.


Introduction
P5, line 100: What species is the following sentence referring to and can the authors please provide a reference?"…matched profiles found in other SARS-CoV-2 infection studies…"

Results
The characterisation of the organoids shows some differences in expression of markers between passages.Can the authors include the passage number used for the infectious work as this may have an influence on other gene and protein expression.
The results of plaque assays and immunohistochemistry provide evidence that the SARS-CoV-2 infection failed to produce infectious virus or infect intact organoids.It would be useful to see where ACE-2 is expressed on these cells and its expression level.The authors mention ACE-2 expression in the Discussion as unpublished observations.This data should be included in the manuscript.
The Introduction states that there are 5 IFNa genes in the JFB genome.This manuscript describes the expression of IFNa4l.Were the primers used to detect this gene able to cross-react with the other 4 IFNa genes?Why was this one chosen for the analysis?
Reviewer #2 (Remarks to the Author): This study presents a rare development and validation of an organoid model in a common bat reservoir host species, the Jamaican fruit bat, and uses this to assess the immune response in vitro to SARS-CoV-2.The results are compelling, particularly given the seemingly strong antiviral response observed in this species (compared to other studies).I do have several suggestions for improvement and some minor minor edits outlined below.L45: I would remove Ebola virus from this list, as bats remain unconfirmed as reservoir hosts for this particular virus.Nipah virus would be a better example with experimental confirmation.L47: I would suggest qualifying this statement slightly as "which is also thought to have its evolutionary origins in bats"; as the authors note in the following sentence, SARS-CoV-2 has not been identified in wild bat populations, only precursor viruses.
L51-56: It may also be worth noting another recent study on wild bat coronaviruses (Becker et al. 2022 Frontiers Virology), in which viruses were relatively common in rectal swabs and also showed little evidence of clinical disease (weak differential abundance of immune proteins via proteomics  Reviewer #3 (Remarks to the Author): The authors established and characterized Jamaican Fruit bat gastrointestinal organoids that recapitulated the organ-specific multicellular composition of the bat gastrointestinal tissue.They demonstrated significantly increased SARS-CoV-2 sgRNA in the bat distal intestinal organoids via qPCR assay but were unable to detect the release of infectious viruses.SARS-CoV-2 infection induced a robust upregulation of interferons and pro-inflammatory genes in the organoid cells.Moreover, SARS-CoV-2 infection of bat organoids led to the activation of cellular regeneration and healing pathways, which might contribute to the improved viral tolerance in this bat species.It is a great endeavor to establish bat organoids for studying the biology underlying bats carrying lots of coronaviruses in a disease-free manner.The strength of organoids over traditional cell lines is that organoids can faithfully simulate the tissues of origin.As such, a meticulous characterization of bat intestinal organoids for recapitulation of bat intestinal epithelium is a prerequisite for these bat organoids for modeling any biological processes.I'd suggest the author clarify the following issues.
1) Characterization of cell types in bat organoids (Fig. 1B). the authors demonstrated the presence of goblet cells by Alcian Blue staining.However, enterocytes are the major cell type in organoids and the native intestinal epithelium.I'd suggest the authors characterize enterocytes within the organoids.
2) The authors showed the stability of the consecutive passage by organoid area and eccentricity in Fig. 1C.Cell-type gene expression profile might be a more favorable assessment to demonstrate the stability of bat intestinal organoids.
3) As far as I know, bats don't have large intestines.The authors may have to verify this in Jamaican fruit bats.If this is the case, authors may amend the terms to proximal and distal intestinal organoids, rather than small intestinal organoids.4) The results of SARS-CoV-2 infection in bat organoids (Fig. 3) were not impressive.After MOI of 1 and 10 inoculation (which is quite high), intracellular viral RNA and those in the culture medium increased more or less over time.Increased sgRNA copy number was observed in 10 MOI infection.The conclusion "JFB organoids were susceptible to SARS-CoV-2 infection" in the abstract, line 31-32, might be insufficiently convincing.

Author response:
We agree with the reviewer that ACE2 expression levels likely have a major impact on susceptibility to SARS-CoV-2 infection.We had previously performed qRT-PCR analysis and now have included ACE2 gene expression data in Fig. 1D and E. Importantly, Ace2 was consistently expressed over multiple passages in the distal intestine, with cT values generally ranging between 27 and 32.Moreover, Ace2 gene expression tended to be higher in the distal intestine than in the proximal intestine or the stomach.However, copy numbers relative to the housekeeping gene GAPDH and compared to other enterocyte genes were low.
We also now performed immunofluorescence analysis of JFB intestinal organoid whole mounts using a crossreactive ACE2 antibody (R&D Systems, AF933), to determine ACE2 protein expression and cellular distribution, As now shown in Fig. 2A, we found weak expression of ACE2 on both the apical and basal cell surface.Conversely, ACE2 protein was not detected in the proteomics screen, which prioritizes highly expressed targets.Based on these results, we conclude that that ACE2 is low but is expected to allow some degree of viral entry.This is now explained in the discussion (lines 301-303).Relevant data are described in the results, lines 140-147 and 153-154.
(4) The Introduction states that there are 5 IFNa genes in the JFB genome.This manuscript describes the expression of IFNa4l.Were the primers used to detect this gene able to cross-react with the other 4 IFNa genes?Why was this one chosen for the analysis?

Author response:
We would like to thank the reviewer for drawing our attention to this issue.To address this question, we have used Primer BLAST to map the primers to the JFB genome.It appears that the Artibeus jamaicensis reference genome in the NCBI database has been updated since our last search and now shows only four protein-coding IFN-α genes: three variants of IFN-α-4-like and one variant of IFN-α-10-like.Importantly, the primers used in our study recognized all four IFN-α gene sequences.We have updated the primer list in the supplemental data, and we refer to Ifna rather than Ifna4l in the results and the figures.We also have revised the description of JFB IFN genes in the introduction (lines 102-103).

Reviewer #2:
(1) This study presents a rare development and validation of an organoid model in a common bat reservoir host species, the Jamaican fruit bat, and uses this to assess the immune response in vitro to SARS-CoV-2.The results are compelling, particularly given the seemingly strong antiviral response observed in this species (compared to other studies).I do have several suggestions for improvement and some minor minor edits outlined below.

Author response:
The manuscript is improved substantially after revision.However, there are still some issues and errors.1.I've mentioned in the initial comments that bats have no large intestines.This is not accurate.I intended to say bats may have no appendix.I apologize for the inconsistency.The authors are suggested to read an earlier paper (Int.J. Morphol., 26(3):591-602, 2008.Comparative Intestinal Histomorphology of Five Species of Phyllostomid Bats).I would also suggest the authors clarify whether the bats (Jamaican Fruit bats) have an appendix or not, and seek assistance from a pathologist to ascertain the nature of intestinal tissues for organoid derivation.2. Line 231-132.As far as I know, goblet cells are an essential cell type in the native intestinal epithelium of both small and large intestines.3. Line 153."Inside-in" conformation is confusing, should be "apical-in".3D organoids maintained in matrigel show an "apical-in" polarity invariably (Viruses.2023 May 14;15(5):1166.Cell Rep. 2019, 26, 2509-2520.e4.), which is not related to any physiology.4. Line 152.Actin is not exclusively present on the apical side of organoids.It is distributed within the cytosol, attached to the cell membrane and enriched on the apical surface.
Reviewer #4 (Remarks to the Author): This study aims to determine the antiviral responses elicited by the GI tract organoids derived from the Jamaican Fruit bat (JFB, Artibeus jamaicensis) challenged with SARS-CoV-2, representing the first study of its kind in a new world bat species.Antiviral responses in bat organoids were determined by qRT-PCR and proteomics (data independent acquisition mass spectrometryuniquely applied to bat organoids).Virus replication was determined by qRT-PCR and the production of infectious virus present in culture supernatant was assessed by plaque assay in VeroE6 cells.Intriguingly, while there was evidence of viral replication, as determined by detection of a modest increase in levels of SARS-CoV-2 nucleic acid, no infectious virus was detected in the culture supernatant.Challenge of organoids with SARS-CoV-2 elicited an increase in the expression of type 1 IFN as well as pro-inflammatory cytokines and chemokines at 48 h, which was not observed with TLR agonists at the same time point, indicating active virus replication is responsible for sustained antiviral and pro-inflammatory responses.Unlike Pteropus alecto, these bat tissues do not appear to demonstrate constitutive expression of type 1 IFN.Analysis of the proteome of SARS-CoV-2 infected organoids revealed that only one ISG, ribonuclease P/MRP protein subunit POP1 (POP1) was significantly upregulated in response to SARS-CoV-2 infection.However, further mechanistic studies to determine the role of this ISG in lack of production of infectious virus was not pursued?Interestingly, SARS-Cov-2 infection led to an increase in organoid size and no evidence of cytopathic effects.Quantitative proteomics analysis suggests activation of innate inflammatory and regenerative pathways upregulated in organoids challenged with SARS-Cov-2.This study reports the generation of GI organoids from JFB and provides novel insights into the interplay between SARS-CoV-2 and the innate immune response in the Jamaican Fruit bat GI organoids and will be of interest to researchers (virologists, immunologists, biologists) studying why bats harbour pathogens without showing clinical signs of disease.

Specific comments (lines refer to marked up version of manuscript):
1. Abstract.Lines 33-34.Suggest adding the following underlined text "…indicating that JFB organoids support only limited viral replication but not viral reproduction" 2. Abstract.Lines 36 -37."gene expression of type I interferon and inflammatory cytokines was induced in response to SARS-CoV-2 but not in response to TLR agonists".This statement is a little misleading since the organoids were responsive to some of the TLR agonists tested with upregulation of gene expression observed at 6 hours -but this effect was not prolonged i.e not observed at 48 -72 hours?Suggest changing to "gene expression of type I interferon and inflammatory cytokines was induced in response to SARS-CoV-2 replication" 3. Line 89 -it would be appropriate to cite the original paper that first reported constitutive expression of IFNalpha in Pteropus alecto i.e -Zhou et al 2016 PNAS doi: 10.1073/pnas.1518240113.

Line 108
The authors emphasise in the introduction that their data is contrary to the "always on" paradigm for antiviral interferon responses to bats.However, it has already been reported in the literature that not all bat species display constitutive expression of antiviral factors (i.e.

L256:
The authors may also consider discussing recent experimental infections of Mexican freetailed bats by SARS-CoV-2 (Bosco-Lauth et al. 2022 Viruses, Hall et al. 2023 Msphere), which even within one species have suggested differing results.L284-288: One possibility the authors should consider in this interpretation (why JFB mount such a strong antiviral response to SARS-CoV-2) is that this subgenus of virus has closely coevolved with Old World bats (especially rhinolophids; Latinne et al. 2020 NatComm) and thus may represent a fairly novel viral challenge.Related work recently on naturally circulating alphacoronaviruses in vampire bats suggested very little proteomic response to infection (Becker et al. 2022 Frontiers Virology), more consistent with the paradigm mentioned earlier by authors.L311: It could also be worth emphasizing the general lack of proteomic approaches in studies of reservoir host immunology (i.e., see Heck & Neely 2020 JPR for a general discussion as well as Hecht-Höger et al. 2020 MolEcology and Neely et al 2021 JPR for rare bat-specific examples, though using wild bat samples and not cell lines).
type 1 IFN in Egyptian fruit bats , Pavlovich et al 2018 Cell; doi: 10.1016/j.cell.2018.03.070.).This should be acknowledged in the introduction and cited. 5. Line 111 Introduction.The authors state that their findings is contrary to the "Always on" paradigm for antiviral interferon responses in bats.See point 4. The authors should temper this statement i.e. "responses in specific bat species" 6. Line 112 -not clear what is meant by "active SARS-CoV-2" -do they mean "infectious SARS-CoV-2"?7. Line 307.The authors state that SARS-Cov-2 genomes were significantly increased in organoid culture supernatant.Are these genomes virion-associated which would suggest the egress of noninfectious virus particles?8. Fig 2D.
Figure legend -define error bars in the graph in the right and the number of independent assays from which these data were derived, which is also needed to justify the statistical analysis.9. Line 323 -the authors state that studies are needed to determine whether JFBs are permissive to SARS-CoV-2 infection in vivo, yet newly added text (lines 285 -287) states that a recent preprint describing in vivo infection experiments in JFBs indicates that it leads to an abortive infection of the intestine without development of clinical disease (ref 66).Please resolve this apparent contradiction.10. "data is not shown" appears in two places ( line 137 and line 186).
Another aspect of novelty that the authors should emphasize here is that all organoids (and most in vitro resources) are in Old World bat species, mirroring more general biases in broader bat virus research (e.g., see a systematic analysis by one of the coauthors, Crowley et al. 2020 Vaccines, as well as a coronavirus-specific analysis by Cohen et al. 2022 bioRxiv).There remains an important need for more research effort (especially for in vitro resources) for New World bats.